〔Abstract〕 Abstract Objective To investigate the selective activation of 5-hydroxytryptamine neurons ( DRN5-HT) in the dorsal raphe nucleus of rats with insomnia induced by p-chlorophenylalanine (PCPA) using chemical genetics tech-niques , and to explore whether it has a regulatory effect on comorbidities of insomnia and anxiety. Methods 32 male SD rats were randomly divided into control group , PCPA model group ( PCPA group) , chemical activation group (hM3Dq + CNO group) , and chemical control group (hM3Dq + Saline group) . The anxiety level of experi- mental rats was evaluated through open field experiments and elevated cross maze experiments . In vivo electroen- cephalography (EEG) was used to record propofol induced sleep and cortical EEG changes , and immunofluores- cence staining was used to observe changes in c-Fos positive protein expression in 5-HT neurons . Results The be- havioral results showed that compared with the chemical control group , the anxiety level of the chemical activation group was significantly lower (P < 0. 05) , and sleep duration was significantly prolonged (P < 0. 05) . The expres- sion level of c-Fos positive protein in DRN5-HT neurons in the chemical activation group was higher (P < 0. 05) . The EEG results showed that the percentage of δ-band power between the chemical activation group and the chemi- cal control group was higher ( P < 0. 05 ) , the power percentage in the α-band was relatively low ( P < 0. 05 ) . Conclusion Chemical activation of 5-HT neurons in the DRN region can improve anxiety levels and increase sleep duration in PCPA induced insomnia rats .